This invention relates to protein-whole microbial cell complexes in membrane form and more particularly to enzymatically active protein-whole microbial cell complex membranes which can be used for catalyzing enzymatic reactions. In another aspect, this invention relates to methods for preparing said enzymatically active membranes and to methods of using said enzymatically active membranes.
2. Description Of The Prior Art
Enzymes are protein catalysts which have been used for a wide variety of industrial and research applications, particularly in pharmaceuticals, paper and textile processing, etc. They are highly specific in their activity and generally do not generate significant quantities of undesirable byproducts. Enzyme reactions are industrially advantageous since they do not require a large investment in heat transfer equipment and can be easily staged, thereby mininmizing the problems associated with interstage product separations.
One problem which has long concerned those dealing with industrial applications of enzymes, however, is the difficulty in separating or recovering enzyme materials. In most commercial processes, the enzymatic rection is effected by simply admixing the enzyme with the substrate, i.e., the chemical being acted upon by the enzyme, and thereafter inactivating and/or recovering the enzyme from the products or the unreacted substrate following the reaction. This procedure, however, has frequently resulted in damage to the product, and inherent loss of large quantities of enzyme, since usually no enzyme is recovered or, if this is attempted, the yields are quite low.
Another problem which has been of significant concern to those engaged in this technology, is that the enzymes usually are used in an aqueous dispersion form. As a rule, however enzymes in this form have a limited shelf life, and, especially, if stored in dilute form, will undergo rapid loss of activity upon storage.
To alleviate these problems, the art has developed various so-called "immobilized enzymes" in which the enzymes are immobilized or bound to inert or insoluble carriers. At the completion of the enzymatic reaction, these insoluble enzyme-containing materials can be separated from the unreacted substrate or product by techniques such as ultrafiltration or the like.
As discussed in Kay, Process Biochemistry, August 1963, which is a synopsis of the history of immobilized enzymes, there have been essentially three known methods of immobilizing an enzyme:
1. covalent bonding onto a chemically modified substrate or onto a substrate capable of covalent bonding; PA1 2. adsorption onto a clay or into a gel, or embedding into an inert matrix (i.e., no bonding); PA1 3. cross-linking the enzyme itself.
All of these prior art techniques, however, have proven to be industrially disadvantageous primarily because the procedures for accomplishing immobilization were complex and required very precise control of pH, temperature, materials and the like.
When an enzyme is chemically modified in preparation for covalent bonding, some of the enzymatically active sites of the enzymes will invariably be chemically attached thereby reducing the activity of enzymes. For instance, Silman et al, Biopolymers, Volume 4, Pages 441-448 (1966), discusses covalently bonding the enzymes to collagen, and p-aminophenylalanine leucine copolymers by use of a diazonium salt. That technique, however, can be shown to produce a product of reduced enzymatic activity.
Adsorption of the enzyme onto a clay or into a gel, or embedding an enzyme into an inert matrix, as taught by Leuschner, British Pat. No. 953,414, also results in products of reduced enzymatic activity, due to the hindrance, or blocking effect, of the matrix.
Cross-linking of an enzyme within itself, such as disclosed by Kay or Silman et al, supra. has similar disadvantages to covalent bonding of enzymes.
In applicant's copending application, Ser. No. 135,753, filed Apr. 20, 1971, applicants had disclosed a new method of immobilizing an enzyme which involved complexing the enzyme with a protein or polypeptide. In one embodiment, it was reported that superior immobilization could be effected simply by casting and thence drying a membrane from a dispersion of protein macromolecules comixed and complexed with an aqueous solution of enzyme
In copending applications Ser. No. 176,546 filed Aug. 31, 1971, applicants disclosed a technique of complexing an enzyme with a protein by electro-codeposition.
It was found that direct bonding of the unmodified enzyme to a protein membrane, such as a membrane of collagen or zein, was possible by a complex form of bonding which involved salt linkages, hydrogen bonding and van der Waal forces, and which resulted in a product of superior activity and stability.
Immobilization of enzymes, however, even by the technique as disclosed in applicant's corresponding application, still entailed certain disadvantages, from the point of view that it was still necessary to first extract the enzymes from whole microbial cells, and then recover the enzymes in a purified form. The available techniques for this extraction and recovery generally result in loss of up to 90% of the enzymes, however, so that it would be desirable to provide a technique which would avoid this substantial disadvantage.
Another difficulty with immobilized enzymes is that they tend to lose much of their activity when subjected to elevated temperatures over extended periods of time. Enzymes are in some cases temperature sensitive to an extent that at temperatures as low as 80.degree.C for times on the order of a few minutes, their enzymatic activity will become rapidly deteriorated while immobilization, in general, seems to have a stabilizing effect on activites. Nevertheless, even immobilized enzymes demonstrate a propensity toward degraded activity over time.
It has been known that whole microbial cells exhibit substantial degrees of enzymatic activity, even without separation and purification of the enzyme components of the cell. Heretofore, however, the only known technique of immobilizing whole microbial cells has been by entrapment in an inert matrix (see Mosbach et al, Biotech and Bioeng, XII, 19 (1970)). The difficulty with that technique, however, is that, as with entrapped enzymes, the matrix tends to have an adverse, or hindering effect on the enzymatic activity of the cell.
Other techniques which had been used for immobilizing enzymes were thought to be inapplicable to whole microbial cells, principally because of the larger size of the cells, usually on the order of microns in characteristic dimensions.
Such large particles cannot easly penetrate into the interstices of an immobilizing membrane, so that it was believed that successful chemical bonding would not be feasible.
In the present invention, it has been found that whole microbial cells can be complexed to a protein or polypeptide in membrane form, and in that form will not only provide a higher degree of enzymatic activity than heretofore attainable in the prior art, but will also exhibit a significantly higher temperature stability due to the protective nature of the cell structure.